1. Field of the Invention
The present invention in the field of biochemistry, immunology and medicine relates to antibodies (“Abs”) or other ligands specific for (a) the binary uPA-uPAR complexes, (b) ternary complexes comprising uPA-uPAR and (c) complexes of uPAR and proteins other than uPA such as integrins. These Abs or non-Ab ligands inhibit the interaction of uPA and uPAR with additional molecules with which the above complexes interact. Such Abs or other non-Ab ligands are used in diagnostic and therapeutic methods, particularly against cancer.
2. Description of the Background Art
A significant body of evidence from studies in vitro and in vivo has established that the urokinase plasminogen activator (uPA) system is central to the process of metastasis, making it a promising target for cancer drug development (Mazar, A P et al. (1999) Angiogenesis 3: 15-32). In addition to uPA, its cell surface receptor (uPAR) is a suitable target for the design and development of cancer therapeutic and diagnostic agents (Mazar, A P (2001) Anti-Cancer Drugs 12: 397-400) because:    (a) uPAR is selectively expressed on metastatic tumor cells and angiogenic endothelial cells (“ECs”), but not on other cells;    (b) uPAR is an important participant in several extracellular and intracellular pathways required for metastasis that are currently the object of intense drug development efforts; and    (c) it is possible to interfere at several different points along the uPA pathway.Thus, uPA and uPAR are promising targets for the development of diagnostics and therapeutics useful against many different types of tumors/cancers.
The uPA/uPAR System and Cancer
Metastasis and angiogenesis share many common functional features that characterize invasive and migratory processes of tumor cells and of ECs. These features include (1) the up-regulation of protease and integrin expression, (2) the loss of cell-cell and cell-matrix contacts, (3) increased responsiveness to growth and differentiation factors, and (4) remodeling of extracellular matrix (ECM) and basement membrane (BasM). All of these contribute to tumor progression.
The uPA “system,” which comprises the serine protease uPA, its receptor uPAR, and its specific serpin inhibitor, plasminogen activator inhibitor-type 1 (PAI-1), plays a central role in many of these activities. The activity of this system is responsible for:                (1) initiating cascades that result in the activation of plasminogen, activating several pro-metalloproteases (proMMPs),        (2) release and processing of latent growth factors such as fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and transforming growth factor-1 (TGFβ),        (3) (a) interactions with components of the ECM such as vitronectin (Vn) and fibronectin (Fn),                    (b) direct interactions with several integrins including α5β1 and αvβ3, and (c) remodeling the BasM and ECM to promote cell motility.Further, the uPA system can also initiate localized fibrin turnover which may play a role in angiogenesis.                        
The expression of uPA and uPAR has been demonstrated in numerous tumor types including glioblastoma, prostate, breast, colon, hepatocellular, and renal cell carcinoma. (Mizukami I F et al. (1994) Clin Immunol and Immunopathol 71:96-104; Hsu D W et al., (1995) Am J Pathol 147:114-23; de Witte J H et al. (1999) Br J Cancer 79:1190-8). The expression of uPA and uPAR are typically greater in more aggressive forms of disease. On tumor cells, this expression is often highest at the invasive front of the tumor. (Buo, L et al., (1995) Human Pathol 26:1133-1138; Yamamoto M et al. (1994) Cancer Res 54:5016-5020). Strong immunohistochemical staining for uPAR in blood vessels associated with the invasive front of breast, colon, and renal cell carcinomas has been reported (Bastholm L et al. Appl Immunohistochem Mol Morphol 7: 39-47; Nakata S et al. (1998) Int. J. Cancer 79:179-186). In the colon carcinoma study, uPAR co-localized with VEGF. The expression of uPA and uPAR has also been observed on tumor-associated macrophages in several tumor types (Ohtani H et al. (1995) Int J Cancer 62:691-6; Xu Y et al. (1997) Hum Pathol 28:206-13). uPA is chemotactic for monocytes and mediates both adhesion and migration of these cells. Adhesion and migration require only uPAR occupancy but not uPA catalytic activity. Thus, the uPA system is believed to contribute to tumor progression by acting on multiple tumor-associated cell types.
Several recent studies have evaluated the therapeutic potential of inhibiting the binding of uPA to uPAR in syngeneic systems. The delivery of an adenovirus-encoded murine amino-terminal fragment of uPA (abbreviated “ATF”—this is the domain of uPA that contains the uPAR binding region) directly into tumors resulted in (a) suppression of neovascularization and (b) arrest of tumor growth (Li H et al. (1998) Gene Ther 5:1105-1113). Due to species “specificity,” murine ATF would be expected to bind only to murine host ECs and leukocytes, not to human tumor cells. This indicates that the tumor inhibition was mediated through the suppression of the host angiogenic response. Finally, a collaborative study between some of the present inventors and S. Rabbani and J. Gladu recently demonstrated that a polyclonal Ab raised against a 100-residue fragment of rat uPAR selectively localized to a rat breast tumor which grew from cells of the Mat BIII cell line (Rabbani S A et al. (2002) Cancer Res 62:2390-97). This polyclonal antibody completely inhibited tumor growth and led to tumor regression.
Unfortunately, despite the promise of targeting the uPA system for therapeutic and diagnostic purposes, research efforts have not resulted in the development of agents suitable for the clinic. Small molecule approaches have been hampered by (1) the difficulty of potently inhibiting a protein-protein interaction (e.g., uPA-uPAR or uPAR-integrin), and (2) the lack of suitable leads or structural information amenable to medicinal chemistry efforts. Several potent peptide inhibitors of the uPA-uPAR interaction have been identified but these would suffer from the typically poor pharmacological properties of peptides and have not demonstrated the requisite levels of activity even in cell-based assays (Ploug M et al. (2001) Biochemistry 40:12157-68).